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Replicating DNA molecules of a deletion mutant of the conjugative R-plasmid R 6 K are cleaved at a single site by the EcoRI restriction endonuclease. Electron microscope examination and measurements of the EcoRI treated replicative intermediate molecules indicate that replication can be initiated at two sites on the plasmid DNA molecule. The two sites are located at about 23 and 39% of total length, respectively, from the EcoRI cleavage site. About 5% of the replicating molecules use both replication initiation sites simultaneously.
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